Interferons (IFNs) are a family of structurally and functionally related proteins that exhibit pleiotropic effects on the growth and function of a variety of cell types. Since their discovery as antiviral agents in 1957, IFNs have been shown to exhibit various potent immunomodulatory effects, including regulation of natural killer cell activity and modulation of major histocompatibility antigen expression, as well as antiproliferative activity against malignant cells (Walter, et at., 1998).
The major classes of IFN are IFN-α, -β, -τ and -ω, which are also designated type I (acid-stable), and IFN-γ (designated as type II, acid-labile). Table 1, below, summarizes the aspects of the major classes of IFNs.
TABLE 1Overview of the InterferonsAspectsType IType IType IType IITypesα & ωβτγProduced by:leukocytefibroblasttrophoblastlymphocyteAntiviral++++Antiproliferative++++Pregnancy Signaling−−+−
The IFN-α family of proteins is now known to consist of at least 14 genes, including one pseudogene and two genes that encode for the same protein. Thus, there are 12 separate IFN-α proteins produced from the 14 genes. The various IFN-α subtypes share approximately 80% identity at the amino acid sequence level.
Interferon alpha-1 (IFNα1), also known as interferon alpha-D (IFNαD), is a type I interferon of wide research and clinical interest. Recent reports have demonstrated the efficacy of recombinant IFNαD in the treatment of various viral diseases in humans as well as in animals (Noisakran and Carr, 2000; Noisakran, et al., 1999). Due to the clinical and research interest in human IFNαD, different expression systems have been developed and employed.
Expression of recombinant human IFNαD (rHuIFNαD) in 1982 was described for a Methylophilus methylotrophus system and an E. coli system which both utilized the lac promoter (De Maeyer, 1982). In 1984, Genentech scientists reported a Saccharomyces cerevisiae system in which the IFNαD gene fused with the x-factor prepro signal sequence yielded a secreted IFNαD protein that had relatively low biological activity (Singh, 1984). Several years later, in 1989, secretory expression of human interferon genes in E. coli and Bacillus subtilis (B. subtilis), using the staphylokinase heterologous expression-secretion signal, was developed (Breitling, 1989). In these studies, only the B. subtilis system, and not the E. coli system, was able to secrete rHuIFNαD into the culture medium. A significant improvement on the intracellular expression of rHuIFNαD in E. coli was reported in 1990 by the use of a defined medium in a fed batch mode during fermentation that allowed for a more efficient expression of the protein at reduced specific growth rates inE. coli However, for over eleven years there has been no additional significant improvements in the production of human IFNαD.
The first IFN-τ to be identified was ovine IFN-τ (OvIFN-τ), as a 18–19 kDa protein. Several isoforms were identified in conceptus (the embryo and surrounding membranes) homogenates (Martal, et al., 1979). Subsequently, a low molecular weight protein released into conceptus culture medium was purified and shown to be both heat labile and susceptible to proteases (Godkin, et al., 1982). OvIFN-τ was originally called ovine trophoblast protein-one (oTP-1) because it was the primary secretory protein initially produced by trophectoderm of the sheep conceptus during the critical period of maternal recognition in sheep. Subsequent experiments have determined that OvIFN-τ is a pregnancy recognition hormone essential for establishment of the physiological response to pregnancy in ruminants, such as sheep and cows (Bazer and Johnson, 1991).
An IFN-τ cDNA obtained by probing a sheep blastocyst library with a synthetic oligonucleotide representing the N-terminal amino acid sequence (Imakawa, et al., 1987) has a predicted amino acid sequence that is 45–55% homologous with IFN-αs from human, mouse, rat and pig and 70% homologous with bovine IFN-αII, now referred to as IFN-Ω. Several cDNA sequences have been reported which may represent different isoforms (Stewart, et al., 1989; Klemann, et al., 1990; and Charlier, M., et al., 1991). All are approximately 1 kb with a 585 base open reading frame that codes for a 23 amino acid leader sequence and a 172 amino acid mature protein. The predicted structure of IFN-τ as a four helical bundle with the amino and carboxyl-termini in apposition further supports its classification as a type I IFN (Jarpe, et al., 1994).
While IFN-τ displays many of the activities classically associated with type I IFNs (see Table 1, above), considerable differences exist between it and the other type I IFNs. The most prominent difference is its role in pregnancy, detailed above. Also different is viral induction. All type I IFNs, except IFN-τ, are induced readily by virus and dsRNA (Roberts, et al., 1992). Induced IFN-α and IFN-β expression is transient, lasting approximately a few hours. In contrast, IFN-τ synthesis, once induced, is maintained over a period of days (Godkin, et al., 1982). On a per-cell basis, 300-fold more IFN-τ is produced than other type I IFNs (Cross and Roberts, 1991).
Other differences may exist in the regulatory regions of the IFN-τ gene. For example, transfection of the human trophoblast cell line JAR with the gene for bovine IFN-τ resulted in antiviral activity while transfection with the bovine IFN-Ω gene did not. This implies unique transacting factors involved in IFN-τ gene expression. Consistent with this is the observation that while the proximal promoter region (from 126 to the transcriptional start site) of IFN-τ is highly homologous to that of IFN-α and IFN-β; the region from −126 to −450 is not homologous and enhances only IFN-τ expression (Cross and Roberts, 1991). Thus, different regulatory factors appear to be involved in IFN-τ expression as compared with the other type I IFNs.
IFN-τ expression may also differ between species. For example, although IFN-τ expression is restricted to a particular stage (primarily days 13–21) of conceptus development in ruminants (Godkin, et al., 1982), preliminary studies suggest that the human form of IFN-τ is constitutively expressed throughout pregnancy (Whaley, et al., 1994).
Significantly, the usefulness of interferons has been limited by the toxicity. Use of interferons in the treatment of cancer and viral disease has resulted in serious side effects. IFN-α was introduced as therapy for chronic hepatitis C in the United States in 1991 and in Japan in 1992 (Saito, et al., 2000). However, use of IFN-α in sufficient dosage to yield clinical efficacy (i.e., at amounts of about 1×106 units/treatment and above) is usually associated with a “flu-like” syndrome characterized by fever, headache, lethargy, arthalgias and myalgias (Tyring, et al., 1992). At doses of 5–10×106 units/treatment and above, other toxicities, such as nausea, vomiting, diarrhea and anorexia, become more frequent. Neuropsychiatric symptoms have also been reported in association with IFN-α treatment (Dieperink, et al., 2000). In addition, some studies suggest that the efficacy of IFN-α treatment is not dose dependent (Saito, et al., 2000), and that treatment with IFN-α is associated with the development or exacerbation of autoimmune disorders in patients with neoplasms or viral hepatitis (Jimenez-Saenz, et al., 2000).
Thus, there exists a need for an interferon protein with high antiviral activity and low cytotoxicity. The present invention is designed to meet these needs.